5-trifluoromethyl derivatives of vitamin b12



Jan. l0, 1961 D. PERLMAN r-:rAL 2,967,860

smz'rFLuoaoMEmyL DEEIVATIVES oF VITAMIN B,a

Filed Aug. 15, 1956 nited States Patent G s-TRIFLUoRoMErHYL DERIVATIVES oF viTAivnN B12 David Perlman, Princeton, and .lohn T. Sheehan, Middlesex, NJ., assignors to lin Mathieson Chemical Corporation, New York, N.Y., a corporation of Virginia Filed Aug. 15, 1956, Ser. No. 604,248

3 Claims. (Cl. 260-211.5)

This invention relates to new cobalamins and, more particularly, to 5-triuoromethybbenzimidazole-hydroxocobalamin and salts thereof [the nomenclature herein is based on Bernhauer et al., Angew. Chemie, 66, 776 (1954)]. Y Y

The new compounds of this invention may be represented by the structural formula in the appended drawing, wherein X is a hydroxy radical or the anion of an, acid, preferably a pharmacologically-acceptable acid. Examples of suitable anions are the anions of the mineral acids (c g., chloride, bromide, sulfate, nitrite, and nitrate), cyanide, cyanate, etc. The compounds of this invention can thus be termed S-triuoromethyl-benzimidazole- X-cobalamins, wherein X represents the anionic radical (e.g., S-triuoromethyl-benzimdazole hydroxo-coabalamm; S-trifluoromethyl-benzimidazole-chloro cobalamin; and 5-triuoromethylbenzimidazole-sulfato-cobalamin). The compounds of this invention are bfologicelly active, and hence can be used in lieu of vitamin B12 (5,6-dimethylbenzimidazole-cyano-cobalamin) in promoting growth of chicks. For this purpose, they are administered in the same manner as vitamin B12 (e.g., as supplements to Chek feeds), the respective dose of the particular 5-triN fluoromethyl-benzimidazole-cobalamin depending on its potency relative tovitamin B12.

To prepare the 5-triuoromethyl-benzimidazole-cyanocobalamin of this invention, Propionibaclerium arabinosum is grown under substantially anaerobic conditions in a suitable nutrient medium containing a precursor such as 2nitro4trifluoromethylaniline, S-trifluoromethylbcnzimidazole, 4-trifiuoromethyl-phenylenediamine-1,2,

or their salts and other derivatives based on the func.

tional amino group, or l-trilluoromethyl-3,4-dinit1'oben zene. The sources of carbon, nitrogen and cobalt, utilizable as media components in this process, are those normally employed in the preparation of vitamin B12. The resultant 5-triuoromethyl-benzimidazole-hydroxocobalamin is then converted, in situ, to S-trifluoromethylbenzimidazole-cyano-cobalamin by treatment of the culture or separated cells (preferably the latter) with potassium cyanide or other cyanide containing salt, and the cyano-cobalamin is isolated as more fully detailed in Example l. The resultant cyano-cobalamin can then be converted to hydroxo-cobalamn and thence to other salts of 5-trifluoromethyl-benzimidazole-cobalarnin by methods known in the art [see Kaczka et al., Jour Amer. Chem. Soc., 73, 3569 (1951)] to yield products which are also biologically active.

The following examples illustrate the invention (all temperatures being in centigrade):

EXAMPLE 1 5-trifluoromethyl-benzmidazole-cyano-cobalamin liters of a medium containing (per liter): glucose, 30 g.; autolyzed yeast, 20 g.; Co(NO3)2.6H2O, 0.01 g.;

5-trifluoromethylbenzimidazole-cyano-cobalamin;

tap water, 1 liter; are placed in a stainless steel fermentation unit of 38 liters capacity, heated at 121 for 30 minutes, and cooled to 30. About 2,000 ml. of a slurry of CaCO3 (containing 600 g. of CaCOS), sterilized by autoclaving, is then added together with l liter of Propionbacterium arabinosum (American Type Culture Collection catalogue number 4965, Washington, DC.) culture grown on this medium for 72 hours in ilasks shaken on a reciprocating shaker (120-1 inch strokes per minutc), located in a 30 constant temperature room. A sterile alcoholic solution of 2-nitro-4-trifluoromethylanlline (sterilized by filtration through a fritted glass filter) containing 1 mg. per ml. is then added so that the final concentration of aniline is 5 mg. per liter (this supplementation being repeated at 24hour intervals), and the culture is allowed to grow in the medium, under virtually anaerobic conditions while being agitated with a turbine mixer rotating at 87 r.p.m. After 72 hours incubation at 30, a sterile solution of glucose is added so that the concentration of the glucose after addition is 30 g. per liter. After 5 days of incubation, the pl-l of the mixture is about 5.3. The fermented medium is then passed through a Sharples Super Centrifuge, or the cells containing 5-tritluoromethyl-benzimidazole-hydroxo cobalamin are separated by iltration through a bed of flter-aid (Celite).

The collected cellsrare resuspended in 3 liters of propanol-water (:20) containing 0.1 g./liter of potassium cyande, and the suspension is heated at 80-90" for 20 minutes. After filtration, the filtrate is concentrated to ab ut 750 ml. The pH is adjusted with 10 N sodium hydroxide to about 7.5, and 25 g. of activated carbon (Darco G60) is added and the mixture stirred. The activated carbon is removed by filtration and eluted with 700 ml. of acetone-water (65:35 v./v.), containing 0.1 g./l ter of potassium cyanide. The acetone is concentrated to 400 ml., and filtered to remove suspended material. After filtration, the filtrate is successively extracted with three equal volumes of phenol-benzene (70 parts 88% phenol-30 parts benzene)- The non-aque ous layer is separated, pooled and diluted with benzene so that the ratio of benzene to phenol is 15: 1. The solution is extracted three times with 200 ml. portions of water, and the aqueous extracts are pooled and concenrated to about 50 ml. The resultant solution is mixed with 1.5 volumes of benzyl alcohol, and the richwater is saturated with ammonium sulfate. The benzyl alcohol layer is separated, and the aqueous layer is reextracted with one-tenth its volume of benzyl alcohol. The rich benzyl alcohol extracts are combined and dried with sodium sulfate and chromatographed on an activated alumina column. The column is washed with 1 part of methanol and 2 parts of acetone, The rich materal is eluted with methanol and the eluate dried under vacuum. The residue is dissolved in a minimum of water and acetone is added until slightly turbid. On standing for Several days, 5-trifluoromethyl-benzimidazole-cyano-cobalamin in the form of red needlelike crystals are recovered. Spectroscopie examination shows maxima at 278, 361, 52,0 and 550 mp.

[Elim 204 (361)] To show the homogenity and activity of the 5-trifluoromethyl-benzimidazole-cyano-cobalamin of this invention, the following tests were conducted. For these tests the cyano-cobalamin was dissolved in water at a concentration of micro-grams of cyano-cobalamin per ml. of water:

TEST I The solution of 5triuoromethyl-benzirnidazole-cyano cobalamin is dried on a iilter paper strip of Whatman 3 MM paper in parallel with samples of 5,6-dimethyl- Patented Jan. 10, 1961l benzimidazole-cyano-cobalamin, adenine cyano-cobalamin, 2-methyl-adenine-cyano-cobalamin, and Fords factor B [Ford et al., Biochem. Jour., 59, 86 (1955)]. The sheet is placed in an ionophoresis apparatus [similar to that described by Holdsworth in Nature, 171, 148 (1953)], and the paper is impregnated with a solution of 0.5 M acetic acid containing 0.02% KCN (w./v.). A potential of about 280 volts is applied for about 17 hours. The sheet is removed and dried. When dry (and free from odor of acetic acid), it is applied for 15 minutes to the surface of an agar plate seeded with a suspension of a vitamin B12-requiring strain of Escherichia coli (ATCC 11105). [The agar medium contains (grams/liter): sucrose, 20 g.; citric acid, 1.2 g.; (NH4)2PO4, 0.4 g.; KCl, 0.08 g.; MgClzHzO, 0.418 g.; MnClEAHzO, 0.036 g.; FeCl3.6H2O, 0.023 g.; ZnCl2, 0.021 g.; CoCl26l-l20, 0.04 g.; agar, 15 g.; triphenyl tetrazolium chloride, 1 g.]. After 18 hours incubation at 37, the agar plate is observed. The positions of zones of growth of the bacteria (noted as red zones due to the reduction of the tetrazolium dye to the colored formazan) are noted in relation to the location on the paper strip where the samples were applied. When the samples from the fermentation supplemented with the 2-nitro-4-triuoromethyl-aniline are analyzed in this way, zones of growth parallel to those obtained when 5,6-dimethylbenzimidazole-cyano cobalamin (neutral ionophoretically), adenine-cyano-cobalamin and factor B are found, with the largest zone in the neutral area.

TE ST II Another aliquot of the solution is applied to a spot about 3 inches from the end of a strip of Whatman No. 1 filter paper parallel to spots of known cobalamins. The chromatogram is developed by the descending method using a solvent mixture containing: 77 ml. of sec.- butanol, 23 ml. H2O, 0.25 ml. KCN solution (5 gms./ml.) and 100 mg. KClOi, for 24 hours (at 25). The strip is dried and applied to the seeded agar plate as above. After incubation, the zones of growth, representing the presence of vitamins of the B12 group (measured with reference to the movement of 5,6-dimethylbenzimidazole cyano-cobalamin), were 0.9, with smaller Zones of growth corresponding to 0.4 (adenine-cyano-cobalamin) and 1.45 (factor B).

TEST III Another aliquot of the solution is applied to a spot about 3 inches from the end of strip of Whatman No. 4 lilter paper parallel to spots of known cobalamins. The chromotogram is developed by the descending method using a solvent mixture containing: see-butanol, 100 ml.; H2O, 50 ml.; KCN [5% solution (w./v.)], 0.25 ml.; acetic acid (glacial), 1.0 ml. After 17 hours development (at 35 C.), the strips are dried and plated on seeded agar plates as above. Zones of growth parallel to those obtained when the 5,6-dimethylbenzimidazolecyano-cobalamin was added are found with smaller zones in the area of mobility about 0.35 and 1.45 that of the 5,6-dimethylbenzimidazole-cyano cobalamin (corresponding to adeninecyanocobalamin and factor B respectivcly) TEST IV Another aliquot is assayed for the presence of substances stimulatlng the growth of Lactobacillus lechmannii (ATCC 7830) using as standard 5,6-dimethylbenthe method of Ford and Porter [Brit. I. Nutrition, 7, 326 (l953)], using the growth response of Ochromanas malhamenss, and the 5,6-dimethylbenzimidazole-cyano-cobalamin as standard; a value of about 0.56 mg. per liter is obtained. When an aliquot from an unsupplemented fermentation is analyzed by these procedures, no growth stimulation of the Ochromonas culture is found (less than 0.05 mg. per liter), and no evidence for production of ionophoretically neutral cobalamins is obtained.

TEST V Another aliquot is assayed for the presence of substances stirnulating the growth of chicks, using the procedure described by Lillie et al. [Poultry Science, 33, 686 (1954)] with replacement of BY500 and bone metal with riboavin and dicalcum phosphate. After 4 weeks growth, the chicks grown on the ration supplemented with the 5-trifiuoromethylbenzimidazole cyano-cobalamn were approximately equal in weight to those grown on the ration supplemented with an equal weight (based on the Lactobacillus lechmanni bioassay) of vitamin Bm.

EXAMPLE 2 5-trfiu0romethyl-benzmidazole-hydroxo-cobalamin (X: OH)

To a solution of 10 mg. of 5-triuoromethyl-benzimidazole-cyano-cobalamin in 10 ml. of water is added 50 mg. of platinum oxide catalyst, and the mixture is shaken with hydrogen at atmospheric pressure for 24 hours. The ltrate from the catalyst is evaporated in vacuo at 25. The residue is dissolved in l ml. of water and 12 ml. of acetone are added. Dark red crystals of 5-triuoromethyl-benzimidazole hydroxo-cobalamin are found on standing for several days. Spectroscopie examination shows maxima at 315, 352, 415 and 530 ma [Elta l EXAMPLE 3 5-triflu0romelhyl-benzmidazole-chloro-cobalamin (X=Cl) 10 mg. of S-triuoromethyl-benzimidazole-hydroxo cobalamin is dissolved in 3 ml. of water and the solution adjusted to pH 4 which 0.1 N hydrochloric acid. The solution is then diluted with l0 ml. of acetone, and after a short time the chloro-cobalamin begins to crystallize in the form of dark red needles. The crystals are separated by centrifuging, Washed with acetone and dried.

Similarly, by substituting hydrobromic acid, sodium nitrite and acetic acid, potassium cyanate and dilute sulfuric acid for the hydrochloric acid in the procedure of Example 3, the corresponding bromo, nitro, cyanato and :ulfato-cobalamins, respectively, are obtained.

The invention may be otherwise variously embodied within the scope of the appended claims.

We claim:

1. A compound selected from the class consisting of 5-triuoromethylbenzimidazole-hydroxo cobalamin and salts thereof.

2. 5-triuoromethyl-benZimidazole-hydroxo-cobalamin.

3. S-triuoromethyl-benzimidazole-cyano-cobalamim References Cited in the tile of this patent UNITED STATES PATENTS 2,561,364 Hall et al. July 24, 1951 2,577,700 CroXall Dec. 4, 1951 2,715,602 Hangrove Aug. 16, 1955 

1. A COMPOUND SELECTED FROM THE CLASS CONSISTING OF 5-TRIFLUOROMETHYLBENZIMIDAZOLE-HYDROXO - COBALAMIN AND SALTS THEREOF. 